1. Field of the Invention(s)
This invention relates generally to protease inhibitors and applications thereof, more specifically to peptide inhibitors of cysteine proteases, even more specifically to allyl sulfones, methods of their use, and methods of their production.
2. Related Art
Protease inhibitors are important therapeutics in the treatment of a variety of disease conditions including viral infections such as HIV infection. Proteases are enzymes that cleave proteins or peptides and are classified into several groups. For example, cysteine proteases form a group of enzymes involved in numerous disease states, and inhibitors of these enzymes can be used therapeutically for the treatment of diseases involving cysteine proteases.
Cysteine Proteases.
Cysteine proteases employ a thiolate residue, which performs a nucleophilic attack on the amide bond of the peptide backbone to form a tetrahedral intermediate. The intermediate collapses to release the first product and the resulting acyl enzyme then undergoes hydrolysis. Based on their sequence homology cysteine proteases are divided into several clans and families. Clan CA and clan CD contain the majority of cysteine proteases. The majority of cysteine proteases, such as papain, calpains, cathepsins, and cruzain belong to the clan CA. According to the crystal structure of papain, clan CA proteases are unique for their catalytic triad formed by Cys, His, and Asn. The oxyanion hole is created by a preceding Gln residue. Clan CA enzymes are inhibited by E-64, a natural inhibitor of cysteine proteases, and cystatin. The substrate specificity of clan CA enzymes is primarily controlled by the S2 enzyme subsite. Clan CD enzymes are unique for their lack of inhibition by E-64 and their specificity for the P1 amino acid residue. Even though clan CD is the smallest of the clans, it contains some very important enzymes. Among them are caspases, legumains, gingipains, clostripain, and separase.
Neural tissues, including brain, are known to possess a large variety of proteases, including at least two calcium-stimulated proteases termed calpains. Calpains are present in many tissues in addition to the brain. Calpain I is activated by micromolar concentrations of calcium while calpain II is activated by millimolar concentrations. In the brain, calpain II is the predominant form, but calpain I is found at synaptic endings and is thought to be the form involved in long term potentiation, synaptic plasticity, and cell death. Other Ca2+ activated cysteine proteases may exist, and the term “calpain” is used to refer to all Ca2+ activated cysteine proteases, including calpain I and calpain II. The terms “calpain I” and “calpain II” are used herein to refer to the micromolar and millimolar activated calpains, respectively, as described above. While calpains degrade a wide variety of protein substrates, cytoskeletal proteins seem to be particularly susceptible to attack. In some cases, the products of the proteolytic digestion of these proteins by calpain are distinctive and persistent over time. Since cytoskeletal proteins are major components of certain types of cells, this provides a simple method of detecting calpain activity in cells and tissues. Activation of calpains and/or accumulation of breakdown products of cytoskeletal elements have been observed in neural tissues of mammals exposed to a wide variety of neurodegenerative diseases and conditions. For example, these phenomena have been observed following ischemia in gerbils and rats, following stroke in humans, following administration of the toxins kainate, trimethyltin, or colchicine in rats, and in human Alzheimer's disease.
Other clan CA proteases, cruzain and rhodesain, are essential for the development and survival of the protozoan parasites Trypansoma cruzi and T. brucei, respectively. T. cruzi causes Chagas' disease in humans in South and Central America, whereas T. brucei causes sleeping sickness in humans in large areas of central and southern Africa. Current drug therapies are accompanied by serious side effects and widespread resistance. Thus, the need of new medicinal agents is urgent.
Cathepsins comprise a large family of lysosomal cysteine proteases and are involved in the degradation of host connective tissues, the generation of bioactive proteins and antigen processing. They have been implicated in a variety of disease states such as rheumatoid arthritis, muscular dystrophy, and tumor metastasis. The high similarity in the substrate specificity among the individual cathepsins presents a challenge in finding selective and thus effective cathepsin inhibitors.
Cysteine Protease Inhibitors.
To date, a structurally diverse variety of cysteine protease inhibitors have been identified. Palmer, (1995) J. Med. Chem., 38, 3193, incorporated herein by reference, discloses certain vinyl sulfones, which act as cysteine protease inhibitors for cathepsins B, L, S, O2 and cruzain. Other classes of compounds, such as aldehydes, nitriles, α-ketocarbonyl compounds, halomethyl ketones, diazomethyl ketones, (acyloxy)methyl ketones, ketomethylsulfonium salts and epoxy succinyl compounds have also been reported to inhibit cysteine proteases. See Palmer, id, and references cited therein. However, most of these known inhibitors are not considered suitable for use as therapeutic agents in animals, especially humans, because they suffer from various shortcomings. These shortcomings include lack of selectivity, cytotoxicity, poor solubility, and overly rapid plasma clearance. Many irreversible cysteine protease inhibitors have been described in the review by Powers, Asgian, Ekici, and James (2002) Chemical Reviews, 102, 4639. See Powers, id, and references cited therein, all of which are incorporated herein by reference.
Among the most effective inhibitors are the vinyl sulfones and α,β-unsaturated carbonyl derivatives. Hanzlik, (1984) J. Med. Chem., 27, 711 has replaced the carbonyl group of a good substrate with a Michael acceptor moiety, which can trap the enzymatic nucleophile (Ser-OH or Cys-OH) without altering the structural features required for enzyme recognition and binding. The fumarate derivative of the epoxy succinate E-64c, which is one of the first Michael acceptor inhibitors reported, extends the α,β-unsaturated carbonyl by an additional carbonyl for possible structural recognition and binding requirements within the enzyme active site. The fumarate derivative of E-64c (trans-HOOCCH═CH—CO-Leu-NH(CH2)2CH(CH3)2) inhibits cathepsin B (kapp=625 M−1s−1), cathepsin H, and cathepsin L (kapp=2272 M−1s−1) irreversibly. Both the fumarate analog of E-64c and the epoxide parent compound do not inhibit clan CD proteases, and are therefore specific for clan CA cysteine proteases. Caspases, legumains, gingipains and clostripain are members of clan CD, while papain, cathepsins, and calpains are members of clan CA. Therefore, because of the aforementioned deficiencies in the art, there is a need for new compounds and methods for inhibiting proteases, in particular cysteine proteases.